Environmental and endogenous exposure to chemicals that produce DNA-protein and DNA-peptide crosslinks are correlated with an increased risk of several cancers, asthma, and other diseases. Currently many thousands of individuals in the US population are exposed to one of the most common DNA-protein crosslink-inducing agents, formaldehyde. These exposures take place in both occupational and in-home settings and affected individuals often experience multi-year chronic exposures that are far in excess of typical indoor air quality standards. Although little is currently known concerning the cellular repair and response mechanisms for this class of DNA lesions, our prior investigations have both rigorously established synthetic chemical procedures to create and utilize DNAs containing site-specifically modified DNA-protein crosslinks and determined dose-dependent, genome-wide assays that identify genes whose products function to limit DNA-protein crosslink-induced cytotoxicity. These investigations have generated a series of hypotheses which postulate that eukaryotic cells exposed to chronic, low levels of DNA-protein crosslinking agents minimize cytotoxicity and mutagenesis through homologous recombination, while following acute high dose exposure, cells will shift to pathways initiated by nuclear proteasome-dependent degradation of covalently linked proteins that can then be processed via either nucleotide excision repair or translesion DNA polymerases. To accomplish our objective of determining the fundamental pathways for the repair and tolerance of DNA-protein crosslinks under chronic and acute exposures, gene-specific deletion analyses will identify the constellation of genes and interrelated pathways that are critical in limiting cellular toxicity and mutagenesis. The roles of individual gene products in modulating cellular response to DNA-protein crosslinks will include repair, recombination, translesion synthesis, cell cycle check points, chromatin remodeling and proteolytic pathways. The activities of translesion synthesis polymerases will be established using DNAs containing site-specific DNA-peptide crosslinks and randomly adducted DPCs. Collectively, these investigations will yield comprehensive analyses of repair and tolerance of this class of lesions